Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy
By Fodale, Valentina; Kegulian, Natalie C.; Verani, Margherita; Cariulo, Cristina; Azzollini, Lucia; Petricca, Lara; Daldin, Manuel; Boggio, Roberto; Padova, Alessandro; Kuhn, Rainer; et al
From PLoS One (2014), 9(12), e112262/1-e112262/31, 31 pp.. Language: English, Database: CAPLUS, DOI:10.1371/journal.pone.0112262
In Huntington’s disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biol. and biophys. properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biol. samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. Methodol./Principal Findings: By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temps., we have discovered a temp.-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. CD spectroscopy confirms the temp. and polyglutamine-dependent change in HTTstructure, revealing an effect of polyglutamine length and of temp. on the alpha-helical content of the protein. Conclusions/Significance: The temp.- and polyglutamine-dependent effects obsd. with TR-FRET on HTT proteins represent a simple, scalable, quant. and sensitive assay to identify genetic and pharmacol. modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington’s disease.
Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor, radiosensitizes human lung and breast cancer cells.
By Bridges Kathleen A; Liu Huifeng; Meyn Raymond E; Toniatti Carlo; Buser Carolyn A; Buchholz Thomas A
From Oncotarget (2014), 5(13), 5076-86, Language: English, Database: MEDLINE
The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.
Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.
By Neuner, Philippe; Peier, Andrea M.; Talamo, Fabio; Ingallinella, Paolo; Lahm, Armin; Barbato, Gaetano; Di Marco, Annalise; Desai, Kunal; Zytko, Karolina; Qian, Ying; et al
From Journal of Peptide Science (2014), 20(1), 7-19. Language: English, Database: CAPLUS, DOI:10.1002/psc.2582
Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. The authors show here that conjugation of NMU to human serum albumin (HSA) yields a compd. with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide deriv. of the peptide with the free thiol of Cys 34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl deriv. of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, the authors’ results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Immunogenicity and therapeutic efficacy of a dual-component genetic cancer vaccine cotargeting carcinoembryonic antigen and HER2/neu in preclinical models.
By Aurisicchio, Luigi; Peruzzi, Daniela; Koo, Gloria; Wei, Wei-Zen; La Monica, Nicola; Ciliberto, Gennaro
From Human Gene Therapy (2014), 25(2), 121-131. Language: English, Database: CAPLUS, DOI:10.1089/hum.2013.103
Several cancer vaccine efforts have been directed to simultaneously cotarget multiple tumor antigens, with the intent to achieve broader immune responses and more effective control of cancer growth. Genetic cancer vaccines based on in vivo muscle electro-gene-transfer of plasmid DNA (DNA-EGT) and adenoviral vectors represent promising modalities to elicit powerful immune responses against tumor-assocd. antigens (TAAs) such as carcinoembryonic antigen (CEA) and human epidermal growth factor receptor-2 (HER2)/neu. Combinations of these modalities of immunization (heterologous prime-boost) can induce superior immune reactions as compared with single-modality vaccines. We have generated a dual component-dual target genetic cancer vaccine consisting of a DNA moiety contg. equal amts. of two plasmids, one encoding the extracellular and transmembrane domains of HER2 (ECD.TM) and the other encoding CEA fused to the B subunit of Escherichia coli heat-labile toxin (LTB), and of an adenoviral subtype 6 dicistronic vector carrying the same two tumor antigens gene constructs. The CEA/HER2 vaccine was tested in two different CEA/HER2 double-transgenic mouse models and in NOD/scid-DR1 mice engrafted with the human immune system. The immune response was measured by enzyme-linked immunospot assay, flow cytometry, and ELISA. The CEA/HER2 vaccine was able to break immune tolerance against both antigens. Induction of a T cell and antibody immune response was detected in immune-tolerant mice. Most importantly, the vaccine was able to slow the growth of HER2/neu+ and CEA+ tumors. A significant T cell response was measured in NOD/scid-DR1 mice engrafted with human cord blood cells. In conclusion, the CEA/HER2 genetic vaccine was immunogenic and able to confer significant therapeutic effects. These data warrant the evaluation of this vaccination strategy in human clin. trials.